Assembly of a Dodecahedral Virus. The assembly mechanism of a virus is critical to its biology, may offer new and untested targets for antivirals, and may be a means to leverage viruses as platforms for nanotechnology. However, assembly is poorly understood. Virus capsids are complexes of tens to thousands of proteins, usually arranged with icosahedral point symmetry. The number of possible assembly intermediates grows combinatorially with the number of units involved in the reaction. Picornaviruses are believed to provide the simplest example of capsid assembly. Based on estimated sedimentation coefficients, it was postulated that picornaviruses are assembled from twelve pentamers. (Dodecahedra share point symmetry with, and are dual to, icosahedra.) We have chosen to examine Bovine Enterovirus (BEV) a non-pathogenic relation of Poliovirus and Human Enterovirus-71. BEV is non-pathogenic even in cattle, where it is endemic. In addition to its ease of handling, in culture BEV produces high yields of 14S intermediates and high yields of empty immature capsid (80S); the presence of these species are what makes it a valuable model for assembly studies. In preliminary data, we establish BEV as a tractable experimental system. We have developed an effective BEV over-expression system. We have accurately measured the molecular weights of 14S and 80S and found they correspond to pentamer and pentagonal dodecahedron, respectively. We have shown that 14S reversibly assembles into 80S. In this proposal, we will define the in vitro assembly mechanism of BEV and use that as a tool for understanding the roles of assembly in BEV replication. In aim 1, we will observe assembly in solution, characterizing assembly energetics and kinetics, fitting kinetics to rigorous mathematical models of assembly, and determining the structures of assembly reactants and products. In aim 2, we will perturb assembly and examine its effect on virus production. Using structures determined in aim 1 and crystal structures from the literature, we will design assembly mutants so that we can compare in vitro activity with mutant activity in culture; we will also examine (for the first time) the effect of small molecules on the picornavirus assembly reaction. These results will contribute an in vitro model of picornavirus assembly and a correlation of assembly biophysics with picornavirus replication. Taken together, these efforts will lead to a far better understanding of the process of virus assembly in vitro and in vivo.